Nucleic acid derivatives such as oligonucleotides appended with various additional functionalities are used widely as research tools in life sciences, in particular, they are regarded as promising therapeutics [1] and sensitive probes for molecular diagnostics [2]. Several oligonucleotide therapeutics have received FDA approval to go into clinic. Examples include an antiviral agent Vitravene (Fomivirsen, ISIS 2922) [3], an anti-angiogenic aptamer Macugen (Pegaptanib sodium) [4] and an anti-cholesterol gapmer Mipomersen (Kynamro, ISIS 301012) [5]. A number of other oligonucleotide candidates such as siRNA, DNAzymes and antisense morpholino analogues (PMO) are currently undergoing various phases of clinical trials.
To be regarded as potential therapeutic candidates, oligonucleotides should correspond to the following requirements.    1. Sufficient stability and sequence-specificity of a complementary complex with their biological target (most often it is a cellular RNA).    2. Increased resistance in biological media such as serum.    3. Beneficial physico-chemical properties such as aqueous solubility and chemical stability.    4. Cost-effective synthesis and affordable price.    5. Efficient cell uptake and in vivo delivery, preferably in the absence of external transfection agents.
According to the mechanism of action, oligonucleotide analogues can interfere with practically any stage of genetic information transfer: either from DNA to RNA (transcription) or from RNA to protein (translation). Inhibition of transcription is performed by binding genomic DNA by triplex-forming oligonucleotides (TFOs) [6], in particular, peptide nucleic acids (PNAs) [7]. Inhibition of translation (antisense mechanism) is realised through mRNA blocking [8]. Most of known to-date oligonucleotide analogues act by antisense mechanism. Those are small interfering RNAs (siRNAs) [9], nucleic acid enzymes (ribozymes or DNAzymes) [10], and a majority of chemically modified oligonucleotide analogues [11]. Specific oligonucleotide derivatives such as aptamers can also block protein function by direct binding to proteins or small molecule co-factors [12].
Most of antisense oligonucleotide analogues bind mRNA and inhibit translation via steric block [13]. Those include a majority of analogues with modifications in the sugar such as 2′-fluoro[14], 2′-O-methyl [15], 2′-O-β-methoxyethyl (2′-MOE) [16] or locked nucleic acid (LNA) [17] derivatives. Oligonucleotide analogues, which substitute an uncharged group for anionic internucleoside phosphate group such as methylphosphonates [18], phosphotriesters [19] or phosphoramidates [20] also act by steric block. The same antisense mechanism is involved in action of distant nucleic acid mimics such as PNAs [21] or phosphorodiamidate morpholino oligonucleotides (PMO) [22].
Additional interest attract those analogues, which are able to irreversibly inactivate RNA by catalysing its hydrolytic cleavage, for example, via recruiting cellular enzyme RNase H by 2′-deoxy phosphorothioates [23], ara-2′-fluoro derivatives (2′-FANA) [24] or gapmers [24]. SiRNAs induce catalytic cleavage of mRNA by activating RISC complex with ribonuclease activity [25] whereas nucleic acid enzymes (ribozymes or DNAzymes) do not require proteins for their catalytic RNA-cleaving action [27].
Many oligonucleotide analogues have modified internucleoside phosphate groups. Among them phosphorothioates [28], phosphorodithioates [29] and boranophosphates [30]. A positive feature of those derivatives is their relatively low cost due to the use of natural 2′-deoxyribonucleotides and highly effective solid-phase DNA phosphoramidite chemistry [31]. Phosphate-modified analogues contain asymmetric phosphorus atom(s) and are usually obtained as a mixture of 2n-1 diastereomers for n-mer oligonucleotide. Different diastereomers often have different affinity to RNA and enzyme resistance, which are crucial for potential antisense action.
Currently, a task of especial priority is the development of oligonucleotide analogues with efficient cell uptake and in vivo delivery, preferably in the absence of external delivery agents such as cationic lipids, polymers or nanoparticles. Here, oligonucleotide analogues with reduced or completely eliminated negative charge may be particularly interesting [32]. Among them are oligonucleoside phosphoramidates that substitute charge neutral phosphoramidate group for anionic phosphate. Chemical synthesis of phosphoramidates is relatively straightforward. However, those analogues exhibit reduced affinity to RNA [33] and are sensitive to acidic hydrolysis [34]. N3′→P5′ phosphoramidates have improved RNA binding but are difficult to synthesise [35]. Those representatives that have positively charged groups in the side chains are more accessible and have excellent enzymatic stability but their affinity to RNA is lower [36]. At the same time a majority of known phosphoramidate derivatives including such useful antisense agents as morpholinos (PMOs) [37] show some lability at acidic pH. Improved acid stability would be required to prevent degradation of oligonucleotide analogues inside endosomes.
Over the last decade new phosphonate oligonucleotide analogues have emerged, which substitute an ionisable phosphonate group for natural phosphate. Among them are phosphonoacetates and thiophosphonoacetates [38], phosphonoformates [39] and 1,2,3-triazol-4-ylphosphonates [40]. Those compounds exhibit increased biological resistance and adequate RNA binding, and, additionally, they support RNase H cleavage and have improved cell uptake even in the absence of transfection agents. However, their chemical synthesis is difficult and costly.
Modified nucleotides and oligonuclides containing at least once the structure P=N-Acc, wherein Acc is an electronic acceptor have also been described [41]. Suitable identities for Acc are —CN, —SO2R and electron-deficient, six membered N+ heterocycles in which at least one nitrogen is alkylated and in an ortho or para position.
At the moment, considerable attention is drawn to phosphorodiamidate morpholino oligonucleotides (PMOs), which are known antisense agents [42]. They are commercially available from GeneTools LLC. PMOs are actively explored as potential therapeutics by Sarepta Therapeutics, (until 2012 AVI Biopharma). In 2013 the company announced successful completion of Phase III clinical trials aganst Duchenne muscular dystrophy (DMD) by a PMO drug Eteplirsen (AVI-4658), which corrects aberrant splicing of dystrophin pre-mRNA [43]. At the beginning of 2014 Sarepta Therapeutics has said that their morpholino drug candidate AVI-7288 has successfully passed Phase I clinical trials against deadly Marburg hemorrhagic fever caused by an RNA-containing virus [44].
However, morpholinos are acid-sensitive just as other phosphoramidates [45]. Moreover, their synthesis is based on P(V) chemistry [46]. The chemistry may lead to side-reactions such as the modification of the O6 in guanine [47]. It can be prevented by a protecting group at the O6 position [48] but that requires a special G monomer, which adds up to the costs of PMO synthesis. Another drawback of the chemistry is that it is incompatible with common phosphoramidite method and cannot use the modifying and labeling reagents available from usual suppliers such as Glen Research, Inc.
Another serious handicap of PMOs is the difficulty of their chemical modification to obtain various derivatives for structure-activity studies. Only a few side-chain modifications to PMO were proposed that are claimed to enhance their cell uptake and therapeutic efficacy [49].
Morpholinos often show relatively poor efficiencies of cell uptake and hence high repeated doses are required for good therapeutic effect to be seen. Cell uptake of PMO-peptide conjugates is much higher than for naked PMO and thus much lower doses are needed in vivo [50]. There is a need to obtain better oligonucleotide analogues that will show greater levels of cell and tissue delivery and improved therapeutic efficacy in the absence of delivery aids.
Thus, the development of new oligonucleotide analogues remains an important task.